Rasayana therapy has been traditionally used in India for its holistic healing systems and proven history of empirical use. There is emerging evidence that Ayurvedic treatment methodologies and herbal medicines may be effective strategies in combating COVID-19. The present study is aimed at evaluating the antiviral and therapeutic activity of an Ayurvedic herbomineral formulation  against the SARS-CoV-2 virus in vitro. A cell-based assay was conducted to evaluate the cytotoxicity of the Svarnvir-IV tablets  for the determination of virucidal activity assessment (at 2 hours) and therapeutic activity assessment . When incubated with SARS-CoV-2 virus at 0.1 multiplicity of infection (MoI) for two hours, Svarnvir-IV tablet exhibited virucidal activity against SARS-CoV-2 with an EC50 value of 0.0058 mg/ml. It also exhibited therapeutic activity when treated with cells infected with the SARS-CoV-2 virus (0.1 MoI) for 1 hour, 2 hours and 4 hours post-infection, with an EC50 value of 0.094 mg/ml, 0.023 mg/ml, and 0.05 mg/ml, respectively. The original supporting data obtained from this study, along with existing Ayurvedic traditional information, has shown encouraging results.The coronavirus disease (COVID-19), caused by the virus SARS-CoV-2, has become a global pandemic in a very short time span. While several vaccines have been developed in the last year, specific treatments for CoV infection are still being explored. Thus, the situation highlights the need to develop safe and efficacious antiviral therapeutics. Ayurvedic  Severe acute respiratory syndrome, caused by a beta coronavirus SARS-CoV-2), is a disease with fever, cough, shortness of breath, and dyspnea. In more severe cases, the infection can cause pneumonia, severe acute respiratory syndrome, kidney failure, and even death. Based on signs and symptoms of COVID-19 as per Ayurveda Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, it may be compared with Ayurveda disease pathology as one of the varieties of Sannipatajajwara  associated with cough [, is a diAs a result, efforts to swiftly develop measures to limit infection rates for the protection of vulnerable people are at an all-time high. In vitro, anti-coronavirus activity has been demonstrated in a growing variety of allopathic medications. However, scientific investigation of antiviral formulations from traditional systems like Ayurveda is yet to be explored.Although Ayurveda is now mostly practiced in India, it had a far broader acceptance and popularity in the past, dating back to the dawn of human civilization and the Vedic period. This system has had numerous ups and downs during its long and illustrious existence. Ayurveda has lately gained international interest due to its significant utilization of plant components, processed minerals, and animal products. Ayurvedic compound formulations are basically divided into two categories:Rasaushadhi: Medicines are prepared predominantly by processed mineral/metallic compounds .Kashtaushadhi: Medicines prepared predominantly by plant drugs.The present study is aimed at evaluating the antiviral and virucidal (irreversible) activity by in vitro testing of an ayurvedic herbomineral formulation  against the SARS-CoV-2 virus to establish its potential as a formulated agent against SARS-CoV-2 infection. The herbomineral product evaluated in this study, the Svarnvir-IV tablet, belongs to the first category, i.e., Rasaushadhi. The study includes an evaluation of cytotoxicity on Vero cells and antiviral testing against the SARS-CoV-2 virus . The study was conducted by ABD-ITC Limited, with the Svarnvir-IV tablet designed by the corresponding author of the study and manufactured by Aimil Pharmaceuticals, New Delhi, India.One of the major constituents of the Svarnvir-IV tablets is Guduchi satva. Other constituents of the Svarnvir-IV tablet are mentioned in Table Adhatoda vasica) is used for the trituration of all ingredients for making tablets and also has therapeutic activity against COVID-19  Determination of the CC50 (drug conc. that reduced cell viability by 50% when compared to the untreated group) and MNTD (maximum non-toxic dose) of the Svarnvir-IV tablet. 2) In vitro antiviral assessment of the Svarnvir-IV tablet-the formulation against the SARS-CoV-2 virus.The in-vitro study was aimed at evaluating the Svarnvir-IV tablet for its potent protease inhibitor activity associated with antiviral activity against SARS-CoV-2. The study protocol was designed to first evaluate the cytotoxicity of the Svarnvir-IV tablet on Vero cells Table . This waTo evaluate the anti-viral (anti-COVID-19) effect of the Svarnvir tablet, a cell-based assay was performed to reveal the cytotoxicity. Further, the determination of virucidal activity assessment (at 2 hours) and therapeutic activity assessment  were performed.Cytotoxicity evaluation: determination of the CC50 and MNTD for the Svarnvir-IV tabletProcedure for MNTD and CC50 determination: Six different dilutions of the test compound are added to the Vero cell monolayer in triplicate. The plates are incubated at 37°C in 5% CO2 for 72 hours. After incubation, a development solution  is added to the cells, followed by incubation at 37°C in 5% CO2. Drug cytotoxicity, CC50, and MNTD are compared with that of 400 μmol formaldehyde as a positive control and diluent development solution (MEM) as a negative control. Absorbance is measured at the appropriate wavelength. MNTD and CC50 are derived.In vitro antiviral assessment of Svarnvir-IV tablet against SARS-CoV-2 virusA) Virucidal activity determination. B) Therapeutic activity determinationVirucidal activity determinationHerein, six different concentrations of the test drug below the MNTD value are assessed for antiviral potency in virucidal mode at the recommended time point in triplicate. Six different concentrations of the test compound in triplicate are incubated with the SARS-CoV-2 virus for 2 hours, recommended contact time. A virus-compound mixture is added to the Vero cell monolayer for infection. The plate is then incubated at 37°C in 5% CO2 for 72 hours. After incubation, the cytopathic effect is compared with hydroxychloroquine sulfate 0.72 μmol as a positive control and MEM as a negative control, and a quantitative CPE (cytopathic effect)-based score is made for EC50 (the compound concentration required to achieve 50% protection from virus) determination-induced cytopathogenicity. CPE stands for cytopathic effect, which is the damage caused to host cells by a virus during infection. When a virus infects a cell, it uses the host's cellular machinery to replicate itself, leading to the destruction of the cell and the release of new virus particles. CPE is a commonly used method for evaluating the antiviral activity of compounds or drugs. In this assay, cells are infected with the virus in the presence of the test compound, and the extent of virus-induced CPE is compared between treated and untreated cells. If the test compound has antiviral activity, it should reduce the severity of CPE compared to the untreated control. The significance of the CPE assay lies in its ability to provide a rapid and reliable measure of antiviral activity. It is relatively simple and inexpensive to perform and can be used to evaluate a wide range of viruses and test compounds. CPE can be visually observed and quantified using various methods, including microscopic examination, automated image analysis, and colorimetric assays.Therapeutic activity determinationSix different concentrations of the test drug below the MNTD value are assessed for antiviral potency in therapeutic mode at recommended time points in triplicate. For each time point, Vero cells are infected with the SARS-CoV-2 virus for the recommended contact time points . Post-infection, six different concentrations of the test compound will be added to infected Vero cells in triplicate. The plate is incubated for 72 hours at 37°C in 5% CO2. After incubation, the cytopathic effect is compared with hydroxychloroquine sulfate 0.72 μmol as a positive control and MEM as a negative control, and a quantitative CPE-based score is made for EC50 determination.Assessment of the drug in all modes will identify the antiviral potential of the drug candidate against SARS-CoV-2. EC50: compound concentration required to achieve 50% protection from virus-induced cytopathogenicity. CC50: compound concentration required to reduce cell viability by 50%. EC80: Drug concentration that gives 80% of Emax (maximum drug effect). SI* (selectivity index): ratio by CC50/EC50.*The higher SI ratio = theoretically more effective and safer drug during in vivo treatment for a given viral infection. The EC50 value is the chemical concentration necessary to provide 50% protection against virus-induced cytopathogenicity. Following incubation, a quantitative CPE-based score will be generated for EC50 determination.For determining the formulation's cytotoxicity, CC50, and MNTD, different dilutions of the test compound were tested on Vero cells and incubated at 37°C in 5% CO2 for 72 hours and compared with 400 µmol of formaldehyde as a positive control and diluent development solution MEM  as a negative control. Similarly, for virucidal activity, hydroxychloroquine sulfates 0.72 µmol as a positive control and MEM as a negative control were used, and other related details are given in Table Svarnvir tablet, when incubated with SARS-CoV-2 virus at 0.1 multiplicity of infection (MoI) for 2 hours, exhibited virucidal activity against SARS-CoV-2 with an EC50 value of 0.0058 mg/ml. It also exhibited therapeutic activity when treated with the cells infected with the SARS-CoV-2 virus (0.1 MoI) for 1 hour, 2 hours, and 4 hours post-infection, with an EC50 value of 0.094mg/ml, 0.023 mg/ml, and 0.05 mg/ml, respectively.The cytotoxicity research on the assessment of Svarnvir-IV reveals that at a dose of 0.500 mg/ml, the CC50 of the Svarnvir-IV tablet is not cytotoxic. The maximum non-toxic dosage, or MNTD, is not included in the statistics that are presented. After two hours, the Svarnvir-IV tablet's EC50 for virucidal activity was determined at a concentration of 0.0058 mg/ml. The EC50 values for therapeutic action were determined at various time intervals: 0.094 mg/ml after an hour, 0.023 mg/ml after two hours, and 0.050 mg/ml after four hours.The selectivity index (SI), which is the ratio of CC50 to EC50, can be calculated as follows: For virucidal activity: SI = CC50/EC50 = 0.500/0.0058 = 86.2; for therapeutic activity after 1 hour: SI = CC50/EC50 = 0.500/0.094 = 5.3; for therapeutic activity after 2 hours: SI = CC50/EC50 = 0.500/0.023 = 21.7; for therapeutic activity after 4 hours: SI = CC50/EC50 = 0.500/0.050 = 10.0.The Svarnvir-IV tablet is anticipated to be more efficient and safer when used in vivo to treat a specific viral infection with a higher SI ratio. Accordingly, the Svarnvir-IV tablet has the highest selectivity index for virucidal action based on the estimated SI values, with therapeutic activity peaking after two hours Table .The data presented in Table The antiviral evaluation of the Svarnvir-IV tablet against the SARS-CoV-2 virus in the virucidal mode is shown in Table The maximum relative percent CPE inhibition was found at a dose of 0.125 mg/ml, indicating that the Svarnvir-IV tablet has antiviral action against the SARS-CoV-2 virus in the virucidal mode. The medication has an EC50 of 0.0058 mg/ml in the virucidal mode, demonstrating its effectiveness in obstructing virus-induced cytopathogenicity. To ascertain the drug's effectiveness in vivo and the ideal therapeutic concentration, more research would be necessary.Svarnvir-IV was also assessed for its antiviral activity against the SARS-CoV-2 virus in therapeutic mode. The relative percent cytopathic effect (CPE) inhibition at various sample concentrations after 1, 2, and 4 hours of treatment was used to gauge the sample's antiviral effectiveness Table . In the Overall, the evidence points to the Svarnvir-IV tablet having antiviral action against the SARS-CoV-2 virus in a therapeutic mode, with the concentration and length of therapy being factors in the reduction of viral reproduction. However, more research is required to ascertain the sample's effectiveness and safety in clinical settings. The development and production of broad-spectrum antiviral medications not only combat COVID-19 but also builds arsenals for future viral epidemics. The richness of Ayurvedic information on virus-host interactions that has been and continues to be acquired will guide and stimulate the development of host-targeting antivirals. These offer the potential benefits of broad-spectrum therapeutic action and viral evasion resistance. Antiviral drug development, in general, will benefit from the increased social knowledge of Ayurvedic medicine and the COVID-19 outbreak, therefore protecting humanity from future viral epidemics. It is found that the antiviral effects of ingredients present in the Svarnvir-IV tablet 450 mg) will block the cell cycle protein of the pathogen and increase innate host immunity. The Svarnvir-IV tablet contains gold microparticles (like nanoparticles) in the absorbable form that inhibit early stages of viral replication, viral entry, and viral attachment by penetrating cells 0 mg will, and a zEndoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation are known to play important roles in viral replication and pathogenesis during coronavirus infection . To sustThe current study was conducted to evaluate the antiviral activity of the Svarnvir-IV tablet against the SARS-CoV-2 virus in vitro. Svarnvir-IV tablet, when incubated with SARS-CoV-2 virus at 0.1 multiplicity of infection (MoI) for 2 hours, exhibited virucidal activity against SARS-CoV-2 with an EC50 value of 0.0058 mg/ml. It also exhibited therapeutic activity when treated with cells infected with the SARS-CoV-2 virus (0.1 MoI) for 1 hour, 2 hours, and 4 hours post-infection, with an EC50 value of 0.094mg/ml, 0.023 mg/ml, and 0.05 mg/ml, respectively. Given the in vitro effectiveness demonstrated in the study, the Svarnvir-IV tablet should be studied further for its potential as an orally administered medication for SARS-CoV-2 therapeutic uses. The original supporting data obtained from this study, along with existing Ayurvedic traditional information, will help to understand the potential of Svarnvir-IV tablets in the treatment of COVID-19.Limitations: The Svarnvir-IV tablet's antiviral activity was assessed in vitro, but more research is needed to determine its safety and effectiveness in both animal models and humans. To fully comprehend the precise mode of action of this medicine, more investigation is also required.Direct-acting antivirals (DAAs) are being created at a rapid rate, and there is a great deal of anticipation that these efforts will result in very effective Ayurvedic antiviral medications. As per this investigation, it is found that the Svarnvir-IV tablet is an inhibitor of the causative virus (SARS-CoV-2) based on the above results. Svarnvir-IV tablet, when incubated with SARS-CoV-2 virus at 0.1 multiplicity of infection (MoI) for 2 hours, exhibited virucidal activity against SARS-CoV-2 with an EC50 value of 0.0058 mg/ml. It also exhibited therapeutic activity when treated with cells infected with the SARS-CoV-2 virus (0.1 MoI) for 1 hour, 2 hours, and 4 hours post-infection, with an EC50 value of 0.094mg/ml, 0.023 mg/ml, and 0.05 mg/ml, respectively. The original supporting data obtained from this study, along with existing Ayurvedic traditional information, will help to understand the potential of the Svarnvir-IV tablet in the treatment of COVID-19. The drug can be rapidly repurposed for safe and highly efficacious clinic applications for treating COVID-19 patients.


Developing modulatory antibodies against G protein-coupled receptors is challenging. In this study, we targeted the follicle-stimulating hormone receptor (FSHR), a significant regulator of reproduction, with variable domains of heavy chain-only antibodies (VHHs). We built two immune VHH libraries and submitted them to multiplexed phage display approaches. We used next-generation sequencing to identify 34 clusters of specifically enriched sequences that were functionally assessed in a primary screen based on a cAMP response element (CRE)-dependent reporter gene assay. In this assay, 23 VHHs displayed negative or positive modulation of FSH-induced responses, suggesting a high success rate of the multiplexed strategy. We then focused on the largest cluster identified  that displayed positive modulation of FSH action. We demonstrated that PRC1 specifically binds to the human FSHR and human FSHR/FSH complex while potentiating FSH-induced cAMP production and Gs recruitment. We conclude that the improved selection strategy reported here is effective for rapidly identifying functionally active VHHs and could be adapted to target other challenging membrane receptors. This study also led to the identification of PRC1, the first potential positive modulator VHH reported for the human FSHR. The follicle-stimulating hormone receptor (FSHR) is a class-A G protein-coupled receptor (GPCR), characterised by a large ectodomain that is responsible for the binding of its cognate ligand, the follicle-stimulating hormone (FSH) . In testThe FSHR is involved in pathologies affecting the reproductive tract such as ovarian cancers , infertiTreatments for assisted reproductive technologies rely on the use of recombinant FSH, as no pharmacological alternative is approved on the market. Recombinant FSH treatment is massively used and is responsible for a list of side effects, including nausea, weight gain, or, more rarely, iatrogenic hyperstimulation syndrome . More reGPCRs are the most abundant family of receptors in the genome and also represent the first class of drug targets . GPCR agParadoxically, despite the tremendous success encountered by antibodies as therapeutic agents, only two GPCR-targeting therapeutic antibodies have been approved to date . VariablIn the present study, we addressed this issue by combining phage-display multiplexing, next-generation sequencing (NGS), and robust functional screening to generate a panel of VHHs potentially modulating the activity of the FSHR. We further characterised one of the VHHs and found that it acts as a potential PAM for the cAMP pathway activated downstream from the FSHR.Lama glama) were immunized using different approaches: one was injected with membranes of HEK293 cells overexpressing the human FSHR; the other was injected in the leg muscle with an expression vector encoding for human FSHR complexed with a nanocarrier. Once transferred into muscle cells, the expression vector led to the expression of FSHR at the cell surface, thereby triggering the immune response. In both cases, leukocytes were isolated from blood and their RNAs were purified. After reverse transcription, VHH repertoires were PCR-amplified and cloned into M13 phagemid vectors. The two immune libraries were subsequently used for phage-display experiments. In order to address the challenge posed by the identification of VHHs capable of specifically modulating the activity of the FSHR, we developed an original selection and screening strategy  the N-terminal peptide (AIELRFVLTKLRVI) located in the FSHR ectodomain.Two llamas  that uses kmer sequence embeddings  and inseTo further explore the potential epitopic diversity covered by the selected candidates, we carried out two unrelated yet complementary in silico analyses.First, we predicted the preferential binding regions on the FSHR ectodomain (ECD) for each candidate using the MAbTope method . Then, tSeveral clusters of similar (ranging from 7 to 2 in size) VHHs were predicted. In this analysis, PRC1 appears to be unique. As expected, the number of clusters was higher in the similarity than in the overlap analysis. We then asked whether or not the predictions made by these two independent in silico methods were congruent. To answer this question, a coherence matrix was computed and revealed a low rate  of incoherencies, PRC1 not being one of them . OverallOf all the potentially interesting VHHs identified in the NGS, functional screening, and epitope prediction, we chose to concentrate on PRC1-Hinge-Fc for further validation characterisation because it displayed a top 3 positive modulation of FSH-induced CRE–luciferase activity while representing, by far, the largest cluster selected, generally a good predictor of favourable affinity.In order to validate the primary screening data, we implemented a procedure that could be amenable to medium-throughput testing quite rapidly. To ensure the parallelisation and cost-effectiveness of the process, the cDNA encoding for PRC1-Hinge-Fc was cloned in a mammalian expression vector (pcDNA3.1) downstream from an IL-2 signal sequence to ensure extracellular secretion. The construct was then transfected in ExpiCHO cells in small volumes (2 mL of medium per well in a 12-well plate). Medium of ExpiCHO cells transfected with empty pcDNA3.1 was used as a negative control. We then assessed the impact of PRC1-Hinge-Fc on the cAMP production profile by bioluminescence resonance energy transfer (BRET) assay, using HEK293 cells expressing the FSHR and the cAMP BRET sensor (NLuc-Epac-vv), in the presence or absence of FSH, with 10 µL of unpurified ExpiCHO supernatant containing PRC1-Hinge-Fc. To further validate PRC1-Hinge-Fc action on FSHR and to explore its pharmacological properties, we produced and purified it in ExpiCHO. on, koff, and KD of PRC1-Hinge-Fc were 3.19 × 104 M−1, 1.59 × 10−3 M−1, and 49.9 nM, respectively. Importantly, these data demonstrate the ability of PRC1-Hinge-Fc to bind the FSH-occupied ECD, confirming an allosteric mode of action.Then, a biolayer interferometry (BLI) assay was carried out to assess PRC1-Hinge-Fc’s ability to interact with the FSHR ECD directly and to determine the kinetic rate constants. Recombinant FSHR ECD bound to FSH was produced, purified, biotinylated, and immobilised on streptavidin sensors. Increasing concentrations of PRC1-Hinge-Fc were then exposed to the sensors to measure the association before being washed to estimate the dissociation B. After Next, we assessed the ability of PRC1-Hinge-Fc to bind to the native form of human FSHR, to cross-react with mouse FSHR and closely related human LHCGR as well as mouse LHR and human TSHR. In order to accomplish this, we carried out flow cytometry analyses. PRC1-Hinge-Fc (300 nM) was applied on cells transfected with cDNA coding human or mouse FSHR, human LHCGR, mouse LHR, or human TSHR. p = 0.043) from 0.63 ± 2.9 nM in the control to 0.15 ± 0.07 nM with PRC1-Hinge-Fc, whereas the Emax increased by 2.4 times . Next, we quantitatively assessed PRC1-Hinge-Fc pharmacological activity on three hallmarks of FSHR signalling: cAMP production, Gs, and β-arrestin recruitment. To this end, three dedicated BRET assays were carried out in living HEK293 cells transiently expressing the hFSHR. First, FSH-induced cAMP responses were compared, with or without the antibody, at different hormone concentrations. Representative kinetics show that PRC1-Hinge-Fc increased cAMP response B, whereap = 0.037), whereas the Emax was unchanged. A significant increase was observed at 3 nM FSH.Next, we carried out a BRET assay to explore the ability of PRC1-Hinge-Fc to modulate the coupling to Gαs. Therefore, we used the Gs sensor miniGs or mGs to assess its recruitment at the FSHR upon hormone stimulation . As showThen, we assessed the effect of PRC1-Hinge-Fc on FSH-induced β-arrestin recruitment to the FSHR using a BRET assay. Even though a slight increase was measured in the presence of PRC1-Hinge-Fc with 3 nM FSH, this did not reach statistical significance and no significant shift was observed in the dose response G–I.Overall, our data establish that PRC1-Hinge-Fc is a PAM for Gs recruitment and cAMP production at the FSHR. The fact that no such effect was observed on β-arrestin recruitment suggests that PRC1-Hinge-Fc could potentially act as a biased allosteric modulator.In this study, we presented an improved method for the selection of modulating VHHs against the FSHR, which belongs to the GPCR family, a particularly important yet challenging class of targets for therapeutic antibodies. Our method is based on the combination of multiplexed phage display and NGS. The idea is that diversifying how the target receptor is exposed/conformed during the selection steps increases the likeliness of obtaining VHHs capable of binding to the native form of the receptor. The drawback of this strategy is that it potentially generates an overwhelming number of clones to assess. In order to circumvent this problem and end up with a tractable number of candidates VHHs, we carried out NGS of all the phage display conditions. Indeed, we found out that randomly picking a limited number of clones, as is usual, often leads to false positives while missing interesting clones. Instead, when analysing millions of NGS sequences and applying very stringent criteria , the total number of candidates dramatically drops. Our data suggest that our strategy leads to identifying hits at a higher rate than with usual VHH selection pipelines.A limitation of relying on NGS for candidate selection is that it requires gene synthesis before testing. We took this as an opportunity to reformat VHHs into VHH-Hinge-Fc and clone them into a mammalian expression vector in frame with a signal sequence to allow for secretion.We reasoned that such reformatting could be beneficial for several reasons: (i) VHH-Hinge-Fc being dimeric benefits from avidity, hence having better apparent affinity; (ii) they present much extended pharmacokinetic properties in vivo thanks to the Fc binding to FcRn , meaningA key advantage of this autologous reporter gene assay is to bypass the bioproduc-tion step, which is tedious and costly. From the 34 candidates selected from the NGS, 23 VHHs had apparent modulatory (negative or positive) effects on the CRE-dependent tran-scriptional activity, suggesting that the multiplexed strategy can favour the selection of candidates with pharmacological diversity. A possible drawback of this method is that antibody concentration in media may vary between candidates, precluding the identification of some interesting candidates. Nevertheless, our procedure allows the rapid detection of positive or negative modulators of receptor activity. Also, it allows the selection of candidates that can be produced efficiently and correctly secreted. To further explore the idea that our strategy can select pharmacologically diverse VHHs, we ran three distinct in silico methods reported to predict the epitopic diversity of antibody sequences. We analysed the germinal lineages of the selected candidates , predicted the epitopes and their overlaps between the different candidates , and computed the sequence and structural similarities of the different candidates. Overall, these in silico analyses suggest that the multiplexed phage-display strategy selects VHHs potentially presenting epitopic diversity. Although there are certainly other factors to consider, one can hypothesise that targeting distinct epitopes on a receptor of interest could favour pharmacological diversity.Here, as an initial step towards validating the multiplexed phage display strategy, we focused on PRC1, which represented the largest sequence cluster by far. Consolidating that choice, a clear functional effect, consistent with a positive modulation of FSHR activity, was found with PRC1-Hinge-Fc in the autologous reporter gene assay and later confirmed using crude ExpiCHO supernatant in a cAMP BRET assay. After that, PRC1-Hinge-Fc was produced and purified, leading to the determination of its binding kinetic rate constants, its binding specificity, and its effect on FSHR signalling. The data demonstrate that PRC1-Hinge-Fc binds specifically to human FSHR with a KD of 49.9 nM. No cross-reactivity was found with the other glycoprotein hormone receptor family members LHCGR and TSHR. Unfortunately, no cross-reactivity with the mouse FSHR was measured, hampering in vivo studies in rodents. Transgenic mice expressing the human FSHR in the FSHR knockout background could help alleviate this important limitation if available in the future. Interestingly, we found that PRC1-Hinge-Fc indifferently binds to the native FSHR without FSH (flow cytometry) and to the FSHR ECD/FSH complex (BLI). We also observed that PRC1-Hinge-Fc modulates the Gs/cAMP pathway only in the presence of FSH and that neither PRC1 nor PRC1-Hinge-Fc interferes with FSH binding to FSHR . Together, these data indicate an allosteric binding mode on the receptor. However, it is important to discriminate allosteric binding from allosteric activity ,30. DemoBased on the recently reported structure of the FSHR in inactive and active conformations , the FSHIn addition, our data revealed no effect of PRC1-Hinge-Fc on FSH-induced β-arrestin 2 recruitment, suggesting that its PAM effect might be biased. Further investigations will be needed to confirm this hypothesis. A study reported binders at the FSHR without any functional evidence being provided . A seconFSHR is a key player in the control of reproduction. Women with loss of function of the receptor experience complete infertility, whereas men display impaired fertility due to sperm’s reduced quality. FSHR is also a potential target for non-hormonal contraception and in ovarian cancers. In addition, the FSH/FSHR axis is increasingly reported to be involved in various pathologies occurring in post-menopausal women. For all these reasons, it is essential to develop a panel of pharmacologically active agents capable of specifically targeting the FSHR to modulate its signalling properties, thereby fine-tuning downstream cellular events and physio-pathological processes. Antibodies in general, particularly VHHs, hold fascinating prospects in that regard. The improved selection strategy we report in this paper could speed up the discovery of more pharmacologically active VHHs, not only for the FSHR but also for other challenging membrane targets.2O at concentrations appropriate for cell stimulation. Ninety-six well white plates from Greiner Bio-one  were used. Coelenterazine-H was from Interchim . Anti-hen egg lysozyme (HEL) VHH was chosen to design the NR control VHH-Hinge-Fc [Recombinant FSH was provided by Merck KGaA  and diluted in mQ HHinge-Fc . Proteolv/v) heat-inactivated foetal bovine serum, 100 IU/mL penicillin, and 0.1 mg/mL streptomycin (ThermoFisher Scientific). Cells were transiently transfected in suspension in 96-well plates with Metafectene Pro  according to the manufacturer’s protocol. ExpiCHO cells  were cultured in ExpiCHO Expression Medium (ThermoFisher Scientific) at 37 °C, 8% CO2, and 120 rpm shaking .HEK293A cells  were cultured in DMEM medium supplemented with 10% (v/v) foetal bovine serum and 0.1 mg/mL streptomycin (ThermoFisher Scientific). Cells were transfected using jetOPTIMUS® according to the manufacturer’s protocol .CHO cells  were cultured in a complete Ham’s F-12K medium supplemented with 10% .Phage display experiments were performed on proteoliposomes , whole cells expressing the FSHR , recombiDNA purified from amplified phages corresponding to each panning experiment were amplified by PCR using VHH-selective primers and sequenced by Next Generation Sequencing (NGS) . The sequences were then grouped into clusters of identical sequences. The sequences had to be found only in the phage display conditions with FSHR and not in the controls.Cricetulus griseus .The VHH candidates’ sequences were reformatted by adding Hinge-Fc from mouse IgG2a containing the LALAPG mutations to suppress the effector functions . FLAG taThe sequences were cloned into the mammalian expression vector pTwist CMV BG WPRE Neo and synthesised by Twist Bioscience . Plasmids were amplified for transfection with NucleoSpin Plasmid Miniprep kit  according to the manufacturer’s protocol.https://forgemia.inra.fr/benoit.piegu/seqcollapse (accessed on 3 July 2023)) to obtain a set of non-redundant sequences and a file describing, for each non-redundant sequence, the number of sequences for each round of selection.NGS sequences were processed using a homemade pipeline written in SnakeMake (unpublished) which carried out the trimming of sequence extremities, their fusion, their translation, and their annotations . The reading frame was determined in the fused sequences by a protein label conserved in the framework 1 (FR1). Sequences were then translated from this frame. After the translation of the sequences, the complementarity-determining regions of the VHHs (CDR) were extracted and concatenated to create the VHH “signature”. The whole CDR sequences were processed with the program seqcollapse (https://forgemia.inra.fr/benoit.piegu/seq2kmermat (accessed on 3 July 2023) was used to encode the sequences from the set of non-redundant sequences as vectors of kmer. From the resulting matrix, a Uniform Manifold Approximate and Projection (UMAP) 36 was created using the R package uwot 37. The different representations of UMAP projection were created using the following parameters: kmer size—5, number of neighbours—15, number of components—2, metric—Euclidean.The program seq2kmermat  minus the epitope overlap. Consequently, a value of 1 corresponds to a pair of antibodies having a similarity of 1  and an overlap of 0 (no overlap of predicted epitopes).We used the FSHR ECD  as the To determine the capacity of the candidate VHHs to modulate CRE-dependent transcription, HEK293A cells were transfected with human FSHR, each VHH-Hinge-Fc to be tested, and the pSomLuc plasmid expressing the firefly luciferase reporter gene under the control of the CRE of the somatostatin promoter region , in DMEM6 cells/mL. Transfections were performed in 1.5 mL ExpiCHO Expression medium in 12-well culture plates (ThermoFisher Scientific), with 0.8 mg/mL of plasmid corresponding to VHH-Hinge-Fc per condition according to the manufacturer’s protocol (ThermoFisher Scientific). The culture was incubated at 37 °C, 8% CO2, and 120 rpm shaking . Sixteen hours after transfection, 5 µL of ExpiCHO Enhancer and 240 µL ExpiCHO Feed (ThermoFisher Scientific) was added per well before putting back the plates at 37 °C, 8% CO2, and 120 rpm shaking. Five days after transfection, 240 µL ExpiCHO Feed was added again in each well and plates were incubated at 32 °C, 5% CO2, with 120 rpm shaking. After 11 to 12 days of transfection, the medium was collected and centrifuged at 4000× g, at 4 °C for 20 min. The supernatants were then collected and stored at –20 °C until further use. The presence of secreted, soluble full-length VHH-Hinge-Fc in the supernatants was confirmed by anti-mouse Western blot.On the day of transfection, ExpiCHO cells (ThermoFisher Scientific) were counted with a LUNA-II cell counter  and density was adjusted to 6 × 10g, 4 °C for 20 min, and the supernatant was dialysed against Tris–NaCl buffer (50 mM Tris–HCl pH 8.0 and 100 mM of NaCl) overnight at 4 °C using 6–8 kDa cellulose membrane tubing (ThermoFisher Scientific). The VHH-Hinge-Fc present in the dialysed supernatant was purified through the capture of the protein A domain present in the Hinge-Fc domain, using UNOsphere SUPrA Affinity Chromatography Media . The protein A beads mixed with the supernatant were incubated for 1 h at room temperature on a rotary wheel. The mixture was then centrifuged and the pellet containing the VHH-Hinge-Fc bound to the beads was put on Poly-Prep® Chromatography Column (Bio-Rad Laboratories). The column was then washed extensively with a washing buffer (50 mM Tris–HCl pH 8.0 and 100 mM of NaCl). The VHH-Hinge-Fc was eluted with a solution of 100 mM of glycine at pH 2.9 and neutralised with a solution of 1 M Tris–HCl at pH 8.0. The eluates were then dialysed against 50 mM Tris–HCl pH 8.0 and 100 mM NaCl. The presence of purified VHH-Hinge-Fc was confirmed by SDS PAGE gel and the preparation was stored at −20 °C until further use.For larger-scale production, ExpiCHO cells were transfected in 30 mL cultures. Ten days after transfection, cell cultures were centrifuged at 4000× Samples were boiled for 10 min at 90 °C in Laemmli buffer. Proteins were separated by SDS-PAGE on 15% polyacrylamide gels that were stained with Coomassie blue.To assess the cAMP production in HEK293 cells, cells were transfected with plasmids coding for human FSHR and an EPAC sensor fused to a nanoluciferase and a tandem acceptor protein, consisting of a circularly permutated Venus and a Venus  .To evaluate Gs protein recruitment, HEK293 cells were transiently transfected with hFSHR C-terminally fused with donor Rluc8 (FSHR-Rluc8) and the miniGs sensor acceptor NES-Venus-mGs  ,41.To measure β-arrestin recruitment, HEK293 cells were co-transfected with FSHR-Rluc8 and a β-arrestin-2 N-terminally fused to a YPET BRET acceptor .−9.5 to 10−6.5 M), 2 µL/well of crude ExpiCHO medium containing the VHH-Hinge-Fc or 1µM of purified VHH-Hinge-Fc diluted in PBS with/containing 5 µM coelenterazine-H. BRET signals were recorded every 60 s for about 60 min in a Mithras LB 943 plate reader (Berthold Technologies GmbH&Co).Forty-eight hours after transfection, cells were stimulated with increasing concentrations of FSH , and a live-dead dye  diluted in staining buffer. Cells were incubated for 30 min at 4 °C and washed twice with staining buffer. The samples were resuspended in PBS and analysed with a MACSQuant Analyser 10 flow cytometer (Miltenyi Biotec). Data were analysed and plotted with FlowJo 7.6.3 .To assess the binding of the candidate VHH-Hinge-Fc to FSHR, CHO cells were transfected with hFSHR, mFSHR, mLHCGR, and hTSHR, which were all synthesised (Twist BioSciences), or hLHCGR . All the receptors had an N-terminal FLAG-tag except hTSHR. Twenty-four hours after transfection, cells were washed and detached with PBS 10 mM EDTA. The cells were mixed with the diluted VHH-Hinge-Fc in a staining buffer  and incubated for an hour at 4 °C. After incubation, cells were washed twice with staining buffer and centrifuged 5 min, 500× ratio. The ECD was loaded at a concentration of 10 nM for 240 s on streptavidin-coated biosensors  before 2 washing steps with PBS 1X-BSA 0.002%. Then, the sensors were incubated for 100 s with increasing concentrations of PRC1-Hinge-Fc and the dissociation was performed in PBS 1X-BSA 0.002% for 150 s. The association/dissociation profiles and the kinetics constants were calculated with an Octet 96 RED interferometer , and the results were analysed with the Octet Data Analysis software (version 9.0).FSHR ECD-Hinge-Fc was produced in ExpiCHO in the presence of FSH and purified according to the protocol described above. The ectodomain was biotinylated using the EZ-link Sulfo-NHS-Biotinylation kit instructions (ThermoFisher Scientific) with a 1:1 biotin:ectodomain To determine if PRC1-Hinge-Fc competed with FSH binding on FSHR, a luminescence assay based on a split luciferase fused to proteins of interest was conducted . FSH wasThe results are shown as mean ± SEM of four or five independent experiments, each performed in triplicate. Data were analysed and plotted using GraphPad Prism 6 .p-values were considered significant at <0.05.The values of all replicates were expressed as the percentage of the maximum of FSH-induced response. Statistical significance was determined by a two-way ANOVA.


The microscopic observations of BNB-MPC dispersions showed less aggregated microstructures and greater structural differences compared with C-MPC dispersions, therefore lowering the viscosity. The viscosity of BNB incorporated (90% amplitude) MPC dispersions at 19% total solids at a shear rate of 100 s−1 significantly decreased to 15.43 mPa·s (C-MPC: 201 mPa·s), a net decrease in viscosity by ~90% with the BNB treatment. The control and BNB incorporated MPC dispersions were spray-dried, and the resultant powders were characterized in terms of powder microstructure and rehydration characteristics. Focused beam reflectance measurement of the BNB-MPC powders indicated higher counts of fine particles (<10 μm) during dissolution, signifying that BNB-MPC powders exhibited better rehydration properties than the C-MPC powders. The enhanced powder rehydration with the BNB incorporation was attributed to the powder microstructure. Overall, reducing the viscosity of feed by BNB incorporation can enhance the performance of the evaporator. This study, therefore, recommends the possibility of using BNB treatment for more efficient drying while improving the functional properties of the resultant MPC powders.Bulk nanobubbles (BNBs) have widespread applications in various fields of science due to numerous peculiar characteristics. Despite significant applications, only limited investigations are available on the application of BNBs in food processing. In the present study, a continuous acoustic cavitation technique was used to generate bulk nanobubbles (BNBs). The aim of this study was to evaluate the influence of BNB incorporation on the processability and spray drying of milk protein concentrate (MPC) dispersions. MPC powders were reconstituted to the desired total solids and incorporated with BNBs using acoustic cavitation as per the experimental design. The control MPC (C-MPC) and BNB-incorporated MPC (BNB-MPC) dispersions were analyzed for rheological, functional, and microstructural properties. The viscosity significantly decreased ( Subsequently, unit operations such as reverse-osmosis/evaporation and spray drying are employed to manufacture MPC in powder form. Simple and innovative strategies before spray drying are of critical consideration for the dairy industry producing MPCs. One of the major concerns for the manufacture of dairy powders is the energy-associated environmental footprint. There were findings that showed that the energy footprint of milk powders is seven times more than packaged milk; therefore, to meet the future need for a significant reduction in energy consumption, innovative technologies are needed ,2. This njection , etc. Renjection .The past two decades have seen substantial industrial and academic importance in investigations of the unique properties of ultrafine bubbles or nanobubbles (NBs). Ordinary bubbles have larger diameters, causing them to rise immediately to the surface of an aqueous solution, collapse or burst out of the solution, and disappear. However, NBs were demonstrated to remain in liquid for an extended period of time . NBs areOne of the key challenges encountered by the MPC manufacturers during the processing is the high viscosity after ultrafiltration and evaporation. Reducing viscosity and aiding an increase in the solid levels before spray drying can offer significant savings on the overall energy cost of powder production. On the other side, it is also a challenge for the end-user to incorporate the MPC powders in a formulation due to their poor rehydration properties, which are influenced by intrinsic powder properties, such as surface and bulk composition, rehydration conditions, storage, etc. . Mechaniw/w) and ultrasonic amplitude  as independent factors were studied. The average protein, moisture, fat, lactose, and ash content of the MPC85 used for this study was 86.05% (w/w), 5.11% (w/w), 0.93% (w/w), 4.81% (w/w), and 6.65% (w/w), respectively. Briefly, MPC85 powders were reconstituted to 15 and 19% TS in DI water by gradual addition of powder using a 4-bladed overhead stirrer  with a temperature-controlled water bath  maintaining the powder dissolution temperature at 40.0 ± 0.1 °C. The overhead stirrer speed was set at 900 rpm during powder addition. Subsequently, the stirrer speed was reduced to 400 rpm. The prepared MPC feed samples were left overnight in a refrigerator to ensure complete rehydration of the powder. Samples were equilibrated to 20 °C in a water bath following overnight storage, and BNBs were incorporated using the continuous acoustic cavitation system as per the experimental design. Control MPC  and BNB-incorporated MPC  dispersions were compared and evaluated for physical, rheological, morphological, and functional properties. In addition, selected combinations  were spray-dried using the spray dryer  to characterize and compare the resultant BNB-MPC powder with the C-MPC powder. The inlet temperature was set at 180 °C, and the outlet temperature ranged between 60 and 65 °C. The spray pressure was maintained at 206.84 kPa, and the relative humidity of the room was periodically recorded using a digital humidity meter . The effect of acoustic cavitation on BNBs were previously reviewed by Bu and Alheshibri, . BNBs weThe control and BNB-incorporated DI water were characterized in terms of size and concentration using the Malvern NanoSight LM10  nanoparticle tracking analysis (NTA) system. NTA a firmly established method that gives real-time visualization, size, and concentration of nanoparticles or NBs in the bulk liquid. All the measurements were performed at 25 °C. The dispersed light from numerous separate nanoscale particles were monitored and the instrument was equipped with a charged-coupled device camera. As a result, the size and relative scattering intensity of each nanoscale particle that scattered light in the water samples were calculated. For each sample, 5 × 60 s videos were recorded, and the parameters used were camera level = 10, threshold = 2. Particles detected by this technique ranged from 10 to 1000 nm in diameter. NTA could not differentiate between the nanoparticles and the BNBs. In addition, the non-existence/traces of titanium nanoparticles were also determined using inductively coupled plasma mass spectrometry. The pure water was incorporated with BNBs and then transferred to the Perkin Elmer NexIon 350 inductive coupled plasma-mass spectrometry for analyzing titanium concentration in bulk NB suspensions. These measurement techniques and their protocols are discussed in more detail elsewhere ,28.−1 to 100 s−1 at 20 °C, and the data obtained were also fitted to the power-law model.The C- and BNB-MPC liquid dispersions were characterized in terms of their rheological properties immediately after incorporation of bubbles. To further understand whether the observed rheological changes were temporary or not, the C- and BNB-MPC liquid dispersions were frozen and analyzed for rheology after thawing. The frozen samples of the C- and BNB-MPC dispersions were thawed in a refrigerator at 4 °C for a day, followed by further thawing in a water bath at 45 °C for 30 min, and were tested for rheological properties. The continuous rotational flow measurement was carried out using a stress–strain-controlled rheometer (MCR-92 Anton Paar) at a varied shear rate from 0.01 sv/v) using DI water before measurements. Proteins were stained using the Fast green FCF  stain. Stock solution of Fast green (5 mg dye in 5 mL water) was applied to the sample for 5–10 min. The stained samples were analyzed in LSM 5 Pa  and were analyzed using the software (LSM-% PASCAL Version 3.2sp2). Three-dimensional images were obtained by scanning the sample across a defined section along the z-axis. Transmission electron microscopy (TEM) was also used to image C- and BNB-MPC dispersions in order to characterize the microstructure. Samples were analyzed for morphology using negative-staining technique. One drop of the diluted sample was mounted on Formvar/carbon-coated 200-mesh copper grids  for 2 min. Subsequently, combined with a drop of 2% uranyl acetate and left for 30 s and the excess liquid was wiped with a filter paper and was examined using a CM 100 TEM  operating at 100 kV, and images captured with a digital camera .The microstructure of C-MPC and BNB-MPC dispersions were studied using confocal laser scanning microscopy (CLSM), following the method described by Gandhi et al. . The micv/v) using DI water before measurements.The average particle size of C- and BNB-MPC dispersions were measured at 20 °C by dynamic laser light scattering using a particle size analyzer  at a scattering angle of 171°. The C- and BNB-MPC dispersions were prepared for particle size analysis by diluting 1:100 (The microstructure of C- and BNB-MPC powders were examined using a scanning electron micrography (SEM) according to the method described by Bouvier et al. . The C- g for 10 min at 25 °C, as described by [The rehydration behavior of the C- and BNB-MPC powder were studied using three techniques: the solubility of the powders was estimated based on the TS in the supernatant obtained by centrifugation at 700× ribed by . Then, tribed by  and elecribed by  methods.w/w) and amplitude  as independent factors for C- and BNB-MPC dispersions and 19% TS with an amplitude of 50 and 90% for C- and BNB-MPC powders. Analysis of variance was carried out using the statistical software SAS  statistical analysis package. The level of significance was set at p < 0.05.Measurements of C- and BNB-MPC dispersion and powder characteristics were performed in triplicate, with results presented as mean ± standard deviation. Data were analyzed as a completely randomized design with TS  DI water, respectively. In other words, the concentration of BNB-incorporated DI water was significantly higher compared to the DI water (~1 billion NBs/mL). One of the common methods that was reported to produce BNBs was based on acoustic cavitation [The control and the BNB-incorporated DI water was characterized in terms of concentration and mean diameter using the nanoparticle tracking system. BNB-incorporated DI water (90% amplitude) had a mean particle size of 235.5 ± 102.3 nm. The particle size distribution of the BNBs was D10, 134.3; D50, 209.2; D90, 369.6 nm. The concentration was found to be 2.41 × 10vitation . NTA andvitation  also usevitation . Notablyp < 0.05) at all the amplitudes  studied. However, it was observed that with the increase in amplitude, the viscosity of MPC dispersions were not significantly (p > 0.05) different from each other. The apparent viscosity of the C-MPC was 68.90 and 201.57 mPa·s at 100 s−1 for 15 and 19% TS, respectively. The viscosity significantly (p < 0.05) decreased to 5.55 and 15.43 mPa·s at 100 s−1 for 15 and 19% TS, respectively, after the BNB incorporation (90% amplitude). A similar reduction in apparent viscosity values were observed after the treatment at 50 and 75% amplitude. This represents a net decrease of ~90% viscosity for BNB-MPC compared to the C-MPC. The observed differences in viscosity could be directly related to particle size and microstructure. A similar trend was reported in a study conducted by Li et al. [n, and the consistency coefficient, κ, for the C- and BNB-MPC dispersions. At 19% TS, the n and κ values were 0.44 and 2.90 Pa Sn for C-MPC, whereas it was 0.74 and 0.06 Pa Sn for the BNB-MPC (90% amplitude) dispersion. Likewise, our previous research on MPC dispersions and Greek-style yogurt also demonstrated the viscosity-lowering effect of BNBs [The general flow characterization of the C- and BNB-MPC dispersions are shown in i et al.  using hy of BNBs ,36. Simi of BNBs  and Song of BNBs  in recon of BNBs  hypothes of BNBs  studied  of BNBs  reported of BNBs . PathaniFurthermore, the freeze–thawed C- and BNB-MPC dispersions exhibited similar behavior to that of the samples tested immediately after the BNB incorporation, and a decreased viscosity was demonstrated at both total solids studied . ResearcThe microstructure of C- and BNB-MPC dispersions (BNB-incorporated powders at 90% amplitude) is shown in The results of particle size analysis of C- and BNB-MPC dispersions are given in 2 and CO2/extrusion porosification, respectively, before spray drying, to produce MPC powders with similar particle structures. Likewise, the BNB incorporation also resulted in MPC powders with these porous structures and the presence of large air voids between the particles. Furthermore, this was also anticipated to facilitate increased water transfer, while also increasing the physical space between casein micelles and was believed to cause a faster structural collapse of the MPC powder particles when added to water [The SEM analysis provided an overview of the shape and size of the C-and BNB-MPC powder particles. Noticeable changes in the microstructure of C-and BNB-MPC powders (19% TS and BNB-incorporated powders at 90% amplitude) were revealed from the SEM analysis . The C-Mto water .2-injected MPC powders (control: 83.6% and N2-treated powders: 96.2%). The more porous structure of BNB-MPC powder particles and the presence of large air voids between these powder particles aided in the enhanced water transfer while also improving the physical space between casein micelles and reducing protein–protein interactions [2-injected and stored (0–180 day) MPC powders. The change in solubility between the control and CO2-treated samples suggests that calcium and other minerals were involved in MPC solubility behavior. In contrast, Li et al. [p < 0.05) different from the BNB-MPC powders. The BNB-incorporated MPC powders at amplitude 50 and 90% had overall mean conductivity of 0.110 ± 0.025 mS/cm and 0.111 ± 0.004 mS/cm (90%), respectively. Furthermore, tomograms were stacked to form a 3D image and the differences were evident form the tomograms. At the end of the 15 min dissolution time, dissolution characteristics for MPC powders were evident and are presented in When comparing the rehydration behavior of the two powders, the BNB-MPC powders showed a much higher solubility index than C-MPC powders. The solubility index of the C-and BNB-MPC powders were 83 ± 0.69, 91.99 ± 0.68 (50% amplitude), and 94.57 ± 0.33% (90% amplitude), respectively. Very recently, McSweeney et al.  observedractions . Similari et al.  used hydi et al.  observedi et al.  reportedi et al.  establis−1 shear rate. Lowering the viscosity of the feed offered the possibility of drying MPC retentates at significantly higher solid concentrations and, thus, more efficient spray drying. Moreover, viscosity had a direct influence on the size of the droplets formed during atomization, reduced viscosity resulted in powder with improved powder properties. Being able to reduce the viscosity of the MPC retentates also results in a reduction in both capital and operational costs. Based on the viscosity data, a 50% amplitude was sufficient to significantly improve the processability of the MPC dispersions; however, during scale-up to meet the spray dryer requirements, this could be further optimized. Notable differences in the microstructure were also observed between the C-MPC and BNB-MPC dispersions subjected to the BNB treatment. Furthermore, the use of BNB treatment offered significant improvements to the rehydration behavior of the MPC powder particles. Thus, this study illustrated how BNB treatment can be employed in MPC processing. Additionally, BNB treatment is an eco-friendly, scalable, and robust process technology.In the present study, we investigated a method for generating BNB using the acoustic cavitation technique. The NTA measurements revealed that bubbles produced in DI water had a mean diameter of 235.5 ± 102.3 nm. The BNB treatment was used as a mechanical pre-treatment to improve the rheological characteristics of MPC dispersions. The BNB treatment of MPC dispersions lowered the viscosity by ~90% at 100 s


T1-weighted spoiled 3D Gradient Recalled Echo pulse sequences, exemplified by Liver Acquisition with Volume Acceleration-flexible MRI (LAVA-Flex), are currently the preferred MR sequence for detecting erosions of the sacroiliac joint (SIJ). However, zero echo time MRI (ZTE) is recently reported to provide excellent visualization of the cortical bone.To directly compare the diagnostic accuracy of ZTE and LAVA-Flex in the detection of structural lesions of the SIJ, including erosions, sclerosis and joint space changes.Two readers independently reviewed the ldCT, ZTE and LAVA-Flex images of 53 patients diagnosed as axSpA and scored the erosions, sclerosis and joint space changes. Sensitivity, specificity and Cohen’s kappa (κ) of ZTE and LAVA-Flex were calculated, while McNemar’s test was employed to compare the two sequences for the positivity of detecting the structural lesions.Analysis of diagnostic accuracy showed a higher sensitivity of ZTE in comparison with LAVA-Flex in the depiction of erosions , especially first-degree erosions (p<0.001) and second-degree erosions (p<0.001), as well as sclerosis , but not joint space changes . Agreement with ldCT was also higher in ZTE in the detection of erosions than LAVA-Flex as indicated by the κ values (0.73 vs 0.47), as well as in the detection of sclerosis (0.92 vs 0.22).With ldCT as the reference standard, ZTE could improve diagnostic accuracy of erosions and sclerosis of the SIJ in patients suspected of axSpA, in comparison with LAVA-Flex. Axial spondyloarthritis (axSpA) belongs to the group of diseases known as inflammatory arthropathies, featuring inflammation in the axial skeleton . Since tConventional MRI sequences such as T1-weighted (T1w) sequence have limited value in the imaging of cortical bone, since the cortical bone has relatively low proton density and presents rapid transverse relaxation of the tissue . CurrentHowever, a number of novel MRI sequences with CT-like bone contrast have emerged ever since. Zero echo time MRI (ZTE) is an exemplary technique for the visualization of osseous structures since it employs zero echo times, enabling the precise depiction of cortical bone contours . PreviouThe objective of this study is to investigate the diagnostic accuracy of ZTE in the detection of structural lesions, including erosions, sclerosis and joint space changes, in the SIJs in patients with axSpA, in comparison with LAVA-Flex using low-dose CT (ldCT) as the reference standard. It is hypothesized that ZTE possessed higher sensitivity and better inter-reader agreement in the detection of bone erosions than T1w 3D-GRE images.This study was a cross-sectional diagnostic study investigating ZTE for the detection of structural lesions in comparison with LAVA-Flex in patients with axSpA. This study was approved by the Ethical Committee of the hospital. Written informed consent was obtained from patients prior to the inclusion of this study ([2020]02-031-01).st, 2021 to January 31st, 2022. Inclusion criteria were diagnosis of axSpA by an expert rheumatologist and fulfillment of ASAS classification criteria for axSpA  with an anterior 30-channel and posterior 40-channel adaptive image receive (AIR) radiofrequency coil. The routine SIJ MRI examination consisted of T2-weighted fat-suppressed turbo spin echo (T2-FS) sequence, T1-weighted images (T1WI) sequence, T1-weighted images with fat saturation (T1-FS) sequence in a semi-coronal orientation and T2-FS sequences in a semi-axial orientation for the SIJ were available. In addition, a ZTE sequence and a LAVA-Flex sequence were performed. Scan parameters of each MRI sequence could be seen in vol) and dose-length product (DLP) were registered from the study protocol for every patient. The mean radiation exposure of ldCT was calculated to be 0.50 mSv (SD 0.07) with a maximum of 0.59 mSv.ldCT was employed as the standard of reference for the detection of structural lesions. All CT examinations were performed on a third-generation dual-source CT . The number of acquired slices was 384 (2×192) slices. Tube voltage was set to Sn100 kV, where Sn indicates the use of a 0.64 mm tin filter, which modifies the spectrum so that exposure to radiation is reduced by blocking low-energy photons . The ldCThe MRI and CT scans were evaluated independently by one radiologist with 9 years’ experience in musculoskeletal imaging evaluation and one rheumatologist with 2 years’ experience in musculoskeletal imaging evaluation. Both observers were blinded to the clinical information and other sequences, with no prior knowledge of the patients’ information. MRI and CT scans were all anonymized and presented to the observers in a random order. Training sessions were assigned to the two observers to score 10 test images in order to reach consensus.Structural lesions, including erosions, sclerosis and joint space changes, were scored based on a method described by Diekhoff et al . with moStatistical analysis was done using R version 3.3. Characteristics of study subjects were summarized with standard descriptive statistics. With ldCT as the standard of reference, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of ZTE and LAVA-Flex were calculated. By using ldCT as the reference standard, the agreement of ZTE and LAVA-Flex with ldCT was evaluated with unweighted Cohen’s Kappa . Such parameters were calculated on the level of quadrants for erosions and sclerosis, while for joint space changes these parameters were calculated on the joint level. A contingency table analysis was conducted comparing ZTE and LAVA-Flex in the detection of structural lesions. The diagnostic performance of the two MRI sequences was compared using McNemar’s test.The sum scores of different structural lesions and diagnostic confidence of ldCT, ZTE and LAVA-Flex were tested for significant differences by Kruskal Wallis’ test. The inter-observer reliability for different structural lesions was assessed with κ value.A p-value less than 0.05 is considered statistically significant.A total of 53 axSpA patients were included in this study. Twenty-three patients were classified as non-radiographic axSpA , and 30 patients were classified as radiographic axSpA . Of the 53 included patients, 34/53 (64.15%) were male and 19/53 (35.85%) were female. The demographic characteristics of the included patients could be seen in The analysis of diagnostic accuracy exhibited that ZTE MRI had higher sensitivity (1582/1710 [92.5%] vs 1393/1710 [81.5%]), accuracy (2391/2544 [94.0%] vs 2202/2544 [86.6%]) and κ value (0.73 vs 0.47) in the detection of erosions on the quadrant level, in comparison with LAVA-Flex, while specificity was comparable (809/834 [97.0%] vs 809/834 [97.0%]) Table 4.The sum scores of different structural lesions with ldCT, ZTE and LAVA-Flex could be seen in The analysis revealed a higher sensitivity (616/680 [90.6%] vs 484/680 [71.2%]), specificity (1848/1864 [99.1%] vs 1073/1864 [57.6%]), accuracy (2464/2544 [96.9%] vs 1557/2544 [61.2%]) and κ value (0.92 vs 0.22) of ZTE in the detection of sclerosis, in comparison with LAVA-Flex Table 4.The median sum scores of sclerosis based on ldCT, ZTE and LAVA-Flex were 6, 6 and 13.5, respectively. The sum scores of sclerosis were not significantly different between ldCT and ZTE (p=0.63), but the sum score of LAVA-Flex is significantly higher than ldCT (p<0.001) and ZTE (p<0.001). The κ values of ldCT and ZTE were 0.82 and 0.83, both indicating very good agreement between the two readers. The κ value of LAVA-Flex was 0.72, suggesting good inter-reader agreement Table 6.Results of the analysis showed a comparable sensitivity (340/357 [95.2%] vs 335/357 [93.8%]), specificity (251/279 [90.0%] vs 238/279 [85.3%]), accuracy (591/636 [92.9%] vs 573/636 [90.1%]) and κ value (0.86 vs 0.79) between ZTE and LAVA-Flex in the detection of joint space changes Table 4.The median sum scores of ldCT, ZTE and LAVA-Flex were 4.5, 5 and 5, respectively. The sum scores of joint space changes were also comparable between each modality. (p=0.973) The κ values of ldCT, ZTE and LAVA-Flex were 0.77, 0.74 and 0.70, respectively Table 6.This study directly compared ZTE and T1w 3D-GRE sequences in the detection of structural lesions in the SIJ. Similar to the previous studies , 8, the By using ldCT as the reference standard, we revealed that ZTE was more accurate than LAVA-Flex in the detection of erosions and sclerosis, but not joint space changes. More specifically, ZTE had an edge over LAVA-Flex in the visualization of low-degree erosions. This advantage could be attributed to the application of zero echo time, enabling the acquisition of sparse MR signal of cortical bone, since cortical bone tissue had a very short T2 relaxation time . ZTE hasPrevious studies by Diekhoff et al. and Baraliakos et al. reported that MR-VIBE had a sensitivity of 95% and 71.2% , while oThe advantage of ZTE over LAVA-Flex in the detection of sclerosis was formidable. Our study reported a sensitivity of 90.6% and a specificity of 99.1% of ZTE in the detection of sclerosis, while the sensitivity and specificity for LAVA-Flex were only 71.2% and 57.6%. Neither of the two previous studies analyzed its capacity in detecting sclerosis . T1w 3D-There are certain limitations to our study. First, the sample size was relatively small with only 53 patients, with no healthy control for ethical consideration that CT could inflict ionizing radiation. Second, the MRI examinations were carried out on a 3.0-T MRI scanner; therefore, our result should not be easily extrapolated to other scanners with different magnetic field strength. Noteworthy, there are several novel MRI sequences with CT-like contrast. The study by Deppe et al. concluded that susceptibility-weighted imaging (SWI) depicts erosions and sclerosis more accurately than T1 spin echo MRI . Jans etIn conclusion, ZTE could generate a near-CT visualization of the SIJ and improve the detection of low-degree erosions and sclerosis in the SIJ with excellent reliability in comparison to T1w 3D-GRE(LAVA). ZTE could be incorporated into the routine MRI examination of the SIJ for patients suspected of axSpA.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethical Committee of the Third Affiliated Hospital of Sun Yat-sen University. The patients/participants provided their written informed consent to participate in this study.Both CL and DL performed the imaging evaluation. CL devised the study design, while DL conducted the statistical analysis and drafted the manuscript. HW was in charge of the acquisition of the imaging data. BL and LT assisted in collecting patient information and helped coordinate this study. JG contributed conceptually to the project and was in charge of the integrity of this study. All authors contributed to the article and approved the submitted version.


To the Editors,1 We agree that it is essential to determine where the right place of an old-school drug is to avoid colectomy during a severe flare-up of UC, considering the novel targeted agents, as well. As most of the data on cyclosporine use in UC comes from the prebiological era, it is necessary to collect as much data as possible to set up an optimal therapeutic order of the current medicines against a severe flare-up. Here we present our experiences on the efficacy and safety of cyclosporine therapy, stratified by prior biologic therapy.We have read with interest the recently published article by Dwadasi et al examining the 1-year colectomy-free survival with the use of calcineurin-inhibitor therapy in severe ulcerative colitis (UC) patients.In our institution, intravenous cyclosporine is used at a dose of 4 mg/bwkg as an induction therapy in severe, steroid-refractory UC for 5 to 7 days followed by oral administration. We retrospectively analysed the short- and long-term data of our hospitalized cyclosporine-treated UC patients, focusing on the previous biological therapy.P < 0.001) and mean C-reactive protein (CRP) level decreased from 66.8 to 15.2 ug/mL (P < 0.001). Reduction of CRP levels and pMayo values did not differ significantly between the group naïve to biological treatment or the group exposed to it.During the same examined period , 27 inpatients received intravenous cyclosporine at our department. Seventy percent of them also received methyl-prednisolone as a rescue therapy, and 75% had pancolitis. Among the patients, 77.8% had prior exposure to anti-TNF and 7.4% to vedolizumab. During the hospitalization, mean partial Mayo score (pMayo) decreased from 7.6 to 2.3 (P = 0.8879). Previous biological treatment(s) did not increase colectomy rate (P = 0.2) in our cohort. Side effect occurred in 1 case as a mild, reversible elevation of serum urea-nitrogen. Prior biologic treatment did not have impact on the efficacy of induction with cyclosporine and did not increase the rate of 1-year colectomy rate. Our data did not support the limited efficacy of third-line cyclosporine; however, we presented a relatively small number of biologic-naïve and third-line patients. Hence, we agree with the authors that further clinical observations are mandatory to determine the optimal sequence of treatment for taking advantage of maximum efficacy and safety.One-year colectomy rate was only 11.1% in our cohort. It should be highlighted that 68% of the patients still received oral cyclosporine at week 52. Duration of cyclosporine use apparently did not have any impact on the rate of colectomy (


Aspergillus fumigatus. However, it is time-consuming and requires expertise. Micronaut-AM (M-AM) is a fast, simple, time-saving, and ready-to-use new colorimetric method using an indicator (resazurin) to facilitate the visual reading. The aim of this retrospective study was to evaluate the performance of the M-AM system and compare it with the EUCAST broth microdilution reference method to determine the susceptibility of 77 A. fumigatus clinical strains to amphotericin B, itraconazole, voriconazole, and posaconazole. Overall, the essential agreements within ±2 dilutions were 100%, 62%, 58%, and 30% and the categorical agreements were 100%, 97%, 91%, and 87% for amphotericin B, itraconazole, voriconazole, and posaconazole, respectively. No categorical discrepancy was found for amphotericin B, but several categorical discordances were observed with azole antifungals. However, only 2 of the 16 azole-resistant strains confirmed by the cyp51A sequencing would have been misclassified by M-AM. The use of M-AM is probably suitable for the determination of the MICs of amphotericin B, but further evaluations are needed to confirm its usefulness for the determination of the MICs of azoles for A. fumigatus.The Antifungal Susceptibility Testing method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST-AFST) is a reference technique for the determination of the Minimum Inhibitory Concentration (MIC) of antifungals for  Aspergillus fumigatus is the most frequent etiological agent responsible for human aspergillosis [Aspergillus-related infections [cyp51A gene [A. fumigatus to azole fungicides in the environment  [cyp51A gene resulting in amino-acid substitutions within the Cyp51A protein  [cyp51A gene associated with the insertion of tandem repeats (TR) in the promoter region (mainly TR34/L98H and TR46/Y121F/T289A) [cyp51A by increasing the binding activity of the sterol regulatory element binding protein, resulting in increased tolerance of azoles [A. fumigatus. Liposomal amphotericin B or a combination of voriconazole and an echinocandin is recommended [gillosis . Triazolfections . Azoles fections . The lan51A gene . Azole rl route) ,5,6. Azond G448) . These mF/T289A) ,8. The pf azoles ,8. The af azoles . Clinicaommended .Early detection of resistance is of paramount importance. It can be performed by different methods, either broth microdilution methods or concentration gradient strip methods. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) broth microdilution methods are the reference techniques for in vitro antifungal susceptibility of filamentous fungi ,12,13. Rcyp51A gene. However, this analysis is difficult to perform routinely, with a delayed response that is not compatible with the treatment of the patient.If the azole-resistance is proven with susceptibility testing, it must be confirmed by molecular analysis. Sanger sequencing is widely used to detect mutations associated with azole resistance in the ®  (M-AM), has been commercialized to determine antifungal susceptibility of Candida spp. and Cryptococcus spp. The ready-to-use plate contains 9 dehydrated antifungals in up to 11 concentrations . Its MICRONAUT-RPMI 1640 medium (including 3-(N-morpholino) propanesulfonic acid (MOPS) and glucose) is used to improve the growth of fungi. The AST indicator (resazurin) added to the test medium facilitates the visual reading [A. fumigatus.Recently, a ready-to-use colorimetric method, the Micronaut-AM Antifungal Agents MIC reading . It is p reading . TherefoA. fumigatus isolates to amphotericin B, itraconazole, voriconazole, and posaconazole.The aim of this study was to compare the M-AM plate and the EUCAST reference method to evaluate the susceptibility of A. fumigatus clinical strains were included in this retrospective study. Isolates were obtained from the samples of 70 hospitalized patients of 2 French hospitals  and stored at −80 °C. After thawing, the strains were cultured on Sabouraud dextrose agar and incubated at 35 °C for 2–5 days to prepare fresh, mature cultures. All of the isolates were A. fumigatus sensu stricto, identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). For all these strains, the azole susceptibility had been tested using the GCS method before storing at −80 °C. The strains with an elevated MIC to azoles  were considered as resistant strains (n = 16). For these strains, the cyp51A gene was secondarily sequenced using the Sanger technique to search for mutations potentially implicated in the azole resistance. The remaining 61 A. fumigatus strains had no elevated MIC to azoles using the GCS method. They were classified as sensible strains. A. fumigatus ATCC 204305 was used as a quality control isolate [Seventy-seven  isolate ,16.6 cfu/mL. The suspension was then 1:1000 diluted into 11.5 mL of MICRONAUT-RPMI-1640 Medium (containing 4% of glucose and MOPS) supplemented with 100 µL of AST indicator (Rezasurin) [3 cfu/mL was distributed into each well with a multi-channel pipette. The plates were incubated at 35 °C. They were read visually under normal laboratory lighting. Rezasurin is an oxidation-reduction indicator. With sufficient fungal metabolism and growth, the oxidized blue indicator switches to the reduced state, which has a pink color [This susceptibility testing was performed by following the M-AM guideline . Brieflyzasurin) . Then, 1nk color . After 2nk color . Only re®, Saint Louis, MO, USA) were amphotericin B, itraconazole, voriconazole, and posaconazole. The range of the concentrations tested was 0.03–16 mg/L for amphotericin B and voriconazole, 0.016–8 mg/L for itraconazole and posaconazole [5 cfu/mL) except for the last column, 12 (used as a negative growth control). The plates were incubated at 35 °C and were read visually with a complete inhibition endpoint after 48 h of incubation [This susceptibility testing was performed by following the EUCAST E.DEF 9.3.2 guideline . The antconazole . Two-folconazole . One huncubation .For each strain, the two methods (M-AM and EUCAST) were performed from the same culture at the same time.cyp51A gene and its promoter region were sequenced. DNA was extracted from the suspension of 0.5 McFarland of the strain using a nucleic acid extraction system . PCR conditions and DNA sequencing were prepared by the method described by Mortensen [cyp51A gene and its promoter region [cyp51A sequence of the A. fumigatus strain AF338659.1 [For the 16 isolates identified as being phenotypically resistant to azole antifungal with the GCS method, the entire ortensen . Eight Pr region . The pro338659.1 .2 dilutions between the two methods. CA is the percentage of isolates interpreted with the two methods in the same category  according to the clinical breakpoint of the EUCAST [Essential agreement (EA) and categorical agreement (CA) were used to investigate the performance of the two methods . The MICe EUCAST . The geoA. fumigatus, the GM of the MICs of amphotericin B was 0.77 mg/L with the EUCAST technique and 0.98 mg/L with the M-AM. The GMs of the MICs with the EUCAST method were 0.46 mg/L, 0.40 mg/L, and 0.20 mg/L for itraconazole, voriconazole, and posaconazole, but were lower  with the M-AM method for these three antifungals  than with the EUCAST technique . The MICs of amphotericin B for this control strain were identical (1 mg/L) with both methods.For the The MICs measured for amphotericin B were within ±2 dilutions in both methods for all isolates, which resulted in a CA and EA of 100%. On the other hand, the M-AM system showed a high CA  but a low EA  for azole antifungals because the MICs measured were always lower with the M-AM method than with the EUCAST technique .For 61 strains, no resistance to azoles and amphotericin B was measured by the GCS method. These strains were also susceptible to amphotericin B, itraconazole, voriconazole, and posaconazole with the two methods (M-AM and EUCAST). cyp51A gene analysis revealed that eight strains had a TR34/L98H alteration and one had a TR46/Y121F/T289A mutation . In the isolate N°1, no mutation was found within the cyp51A gene, so it was considered to be wild type (WT)  . It shoucyp51A gene. Therefore, an isolate was considered as resistant or ATU if it was categorized resistant or ATU to at least one azole drug . Fourteen strains were identified to be phenotypically azole-resistant and two strains to be ATU with the EUCAST method. Eleven strains were identified to be phenotypically azole-resistant, three strains to be ATU, and two strains to be S with the M-AM method . Only 1/16 strain (N°10 TR46/Y121F/T289A) was categorized S with both methods.The most important discordances were found with posaconazole. Twelve categorical discrepancies were found: seven very major (R with the EUCAST method and S with the M-AM method) mainly with the strains with the TR34/L98H mutation (5/7) and five minor . Moreover, two strains  were susceptible with both techniques .A. fumigatus to amphotericin B, itraconazole, voriconazole, and posaconazole.The objective of this study was to evaluate the performance of the M-AM system by comparing it with the EUCAST broth microdilution reference method to determine the susceptibility of 2 dilution. As a result, the overall CA between the two methods was found to be high for amphotericin B (100%), for itraconazole (97%), for voriconazole (91%) and to be moderate for posaconazole (87%). A study published by Nuh et al. evaluated the M-AM system by comparing it with the reference CLSI technique for 78 clinical isolates of the Aspergillus species [Globally, the MICs of itraconazole, voriconazole, and posaconazole using M-AM were always lower than with EUCAST. The GM of MICs obtained by M-AM were approximately three-fold dilution lower for voriconazole and posaconazole, and two-fold dilution lower for itraconazole than those observed using the EUCAST method. For amphotericin B, the MICs obtained by the two methods were almost identical, within ±1 log species . They fo species . The EA  species . It is p34/L98H and N°14 G54E). These two strains had intermediate MIC values with EUCAST, suggesting a weak expression of the resistance in vitro. Furthermore, there were three isolates  categorized to be ATU with M-AM but resistant with EUCAST. The MIC values of itraconazole of these strains (2 mg/L) with the M-AM method suggest that they could be azole-resistant isolates, even though they are susceptible to posaconazole and voriconazole. In this case, it is necessary to perform additional tests such as the cyp51A sequencing or other antifungal susceptibility testing in order not to miss an isolate potentially resistant to azoles.However, the M-AM assay allowed the identification of azole resistance for all resistant isolates except for two  [The EUCAST method is very difficult to use as a routine technique. Therefore, we were very interested to know whether M-AM could be used for the susceptibility screening of by M-AM) .3 cfu/mL with the M-AM method and 100-fold more concentrated with the EUCAST technique 2–5 × 105 cfu/mL [The reasons for the discrepancies of the azole MICs between the two methods (M-AM and EUCAST) are not clear but there are a few hypotheses. For the reference method for filamentous fungi published by the Clinical Laboratory Standard Institute (CLSI), it is well known that the size of inoculum, the type of growth medium, the time of incubation, and the inoculum preparation method can influence MIC values . Several5 cfu/mL ,15. More5 cfu/mL . Therefo® , etc.) could influence the MIC values [34/L98H. They found nine categorical discrepancies (eight minor errors and one strain interpreted resistant by the EUCAST versus susceptible by the GCS) [® ) with CLSI found also very major discrepancies  in three A. fumigatus with the mutation TR34/L98H [On a broader scale, the culture conditions and the reading of the different antifungal susceptibility techniques  . AnotherR34/L98H . These rcyp51A sequencing) to confirm the resistance to azoles. In our study, none of the susceptible strains and only two of sixteen azole-resistant strains (categorized S instead of ATU) would have been wrongly categorized with M-AM. No very major discrepancy was detected by using the global azole categorization. Moreover, for the first isolate (N°1), no mutation was found in the cyp51A gene whereas the two methods detected an elevated MIC for the itraconazole (8 mg/L with the EUCAST method and 2 mg/L for the M-AM method), suggesting that another mechanism of azole resistance was involved. Therefore, it is advised to analyze the results according to the interpretation of the three azoles  in order to identify a resistant strain. Itraconazole seems to be the most suitable drug for the resistance screening because itraconazole resistance was detected by M-AM for most strains (10 of 16 strains). The supplementing of M-AM plate with isavuconazole might increase the possibility of detecting azole resistance.In the routine clinical laboratory, it is important to use an in vitro antifungal susceptibility assay that can detect the isolates resistant to at least one azole drug. These strains could then be further studied with other techniques (determination of MICs by the reference EUCAST method, the cyp51A sequencing and the MICs obtained with the M-AM system. We were not able to do this in our study because the number of strains tested is not large enough.Furthermore, we used the clinical breakpoints of EUCAST suggested by the M-AM manufacturer’s protocol to categorize the isolates. If it is confirmed that MICs measured by this method are always lower than those obtained with EUCAST, these breakpoints should probably be revised on the basis of the A. fumigatus. Therefore, more studies are needed to confirm the possibility of using M-AM for the screening of the susceptibility of A. fumigatus to azole antifungals in routine laboratories.In conclusion, the use of the M-AM technique is probably suitable for the determination of the MICs of amphotericin B, although it must be confirmed by the evaluation of amphotericin B-resistant strains. However, our findings show that this new colorimetric method presents limits for the evaluation of the azole resistance of


The existence of an interconnected mechanism between cognitive disorders and periodontitis has been confirmed by mounting evidence. However, the role of age or sex differences in this mechanism has been less studied. This study aims to investigate sex and age differences in the characterization of periodontal bone tissue, immune state and cognitive function in amyloid precursor protein/presenilin 1(APP/PS1) murine model of Alzheimer’s disease (AD).Three- and twelve-month-old male and female APP/PS1 transgenic mice and wild-type (WT) littermates were used in this study. The Morris water maze (MWM) was used to assess cognitive function. The bone microarchitecture of the posterior maxillary alveolar bone was evaluated by microcomputed tomography (micro-CT). Pathological changes in periodontal bone tissue were observed by histological chemistry. The proportions of helper T cells1 (Th1), Th2, Th17 and regulatory T cells (Tregs) in the peripheral blood mononuclear cells (PBMCs) and brain samples were assessed by flow cytometry.The learning ability and spatial memory of 12-month-old APP/PS1 mice was severely damaged. The changes in cognitive function were only correlated with age and genotype, regardless of sex. The 12-month-old APP/PS1 female mice exhibited markedly periodontal bone degeneration, evidenced by the decreased bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and bone mineral density (BMD), and the increased trabecular separation (Tb.Sp). The altered periodontal bone microarchitecture was associated with genotype, age and females. The flow cytometry data showed the increased Th1 and Th17 cells and the decreased Th2 cells in the brain and PBMC samples of 12-month-old APP/PS1 mice, compared to age- and sex-matched WT mice. However, there was no statistical correlation between age or sex and this immune state.Our data emphasize that age and sex are important variables to consider in evaluating periodontal bone tissue of APP/PS1 mice, and the cognitive impairment is more related to age. In addition, immune dysregulation  was found in the brain tissue and PBMCs of APP/PS1 mice, but this alteration of immune state was not statistically correlated with sex or age.The online version contains supplementary material available at 10.1186/s12974-023-02790-1. Periodontitis is a chronic inflammatory disease characterized by the destruction of the periodontium . Recent P. gingivalis-LPS, one of the main virulence factors of P. gingivalis, led to learning and memory impairment in mice with intraperitoneal injection and rats with injection in the gingival sulcus [Alzheimer’s disease (AD) is the most common form of dementia. It is a multifactorial progressive degenerative disorder that mainly affects aged people. Numerous epidemiological investigations and clinical observations have shown that periodontal infection might damage the central nervous system . In our l sulcus . However+T cells can differentiate into multiple subtypes, including helper T cells1 (Th1), Th2, Th17 and regulatory T cells (Tregs) [Imbalance in immune homeostasis is a common causative factor in periodontitis and AD . In the  (Tregs) . They al (Tregs) . The bal (Tregs) . Machhi  (Tregs)  reportedMultiple studies have demonstrated sex differences in the prevalence, risk, and severity of AD patients . Both ep+ T subsets in the blood and brain were assessed by flow cytometry. This study aims to investigate sex and age differences in the characterization of periodontal bone tissue, immune state and cognitive function in APP/PS1 murine model of AD. In addition, this study may reveal the damage caused by AD to the periodontium and its associated mechanisms of immune imbalance.In the present study, we report data from comparative studies using the Morris water maze (MWM) to assess spatial memory function in APP/PS1 mice of different sexes at 3 and 12 months of age. Histological assessment and micro-CT of the maxilla were performed upon completion of behavioral testing. The changes in the proportion of CD4Specific pathogen-free (SPF), male and female mice were used in the study. We used 3- and 12-month-old APP/PS1-mutant transgenic mice and age-matched nontransgenic wild-type (WT) littermates in this work. All animals were obtained from the Nanjing University Institute of Biomedical Sciences . The transgenes were confirmed by PCR genotyping of mouse tail tissue. All animal experimental protocols were approved by the ethics committee of Animal Care and Experimental Committee of Ninth People’s Hospital affiliated to Shanghai Jiao Tong University of Medicine (SH9H-2019-A499-1) and were carried out under the Guidelines from the EU Directive 2010/63/EU.n = 5, per group). The experimental animals were housed in a temperature- and humidity-controlled room under a 12-h light/dark cycle and were given free access to food and water. Prior to commencement of the experiments, the mice were allowed to acclimatize to their environment for 1 week. After the MWM tests were performed, all mice were sacrificed with sodium pentobarbital  according to the Panel on Euthanasia of the American Veterinary Medical Association. Maxillary bone was dissected, and then analyzed by microcomputed tomography (micro-CT) and histological analysis. Brain tissues and PBMCs were collected for flow cytometry.The mice were divided into two age subgroups: 3 months of age and 12 months of age. The APP/PS1 mice in each subgroup were divided into male and female groups. Age- and sex-matched littermate WT mice were used as controls  consisted of a black circular pool (with a diameter of 120 cm and a depth of 50 cm), a black platform (9 cm in diameter) and a video analysis system. The pool was filled with water until the platform in the pool was submerged 1 cm below the surface. The water temperature was held at 22 ± 1 ℃. The MWM test was performed in a dark room. The mice were released randomly with their heads facing toward the pool wall. They were given 90 s to learn to use the visual tips around the pool to find the hidden platforms. If the mouse failed to find the platform within 90 s, it was guided to the platform and allowed to stay there for 30 s [Each mouse was trained for 4 times per day, and each training interval was 30 s. During the training, the time to find the platform was recorded as the latency. The probe trial was carried out on the sixth day to estimate retention performance. The platform was removed and the mice performed two trials in which they were placed in the two quadrants away from the platform and allowed to swim for 60 s . The num−1), trabecular thickness , trabecular separation  and bone mineral density  were analyzed to evaluate bone quality and resorption. The region of interest (ROI) was selected in the maxillary first molar area.The excised maxillae were placed with gauze in the sample holder and scanned with a benchtop micro-CT imager  at 9 μm resolution, 49 kV, and 179 μA. Bundled vendor software (CTAN software) was used for three-dimensional reconstruction and data processing. The measured structural parameters including bone volume/tissue volume , trabecular number , which was replaced every 3 days for 1 month, dehydrated, and then embedded in paraffin. Maxillary sections were made from the mesio-distal direction. Serial sections of 4 μm in thickness were cut. A hematoxylin–eosin (H&E) staining assay was used in accordance with the manufacturer’s instructions .l-glutamine at 37 ℃ in a 5% CO2 environment. To stain for the intracellular cytokines IFN-γ, IL-4, and IL-17A, cell suspensions were stimulated for 5 h in the presence of GolgiPlug . The cells were fixed, permeabilized, and stained according to the manufacturer’s instructions at 4 °C in the dark, and the data were analyzed by NovoExpress V1.4.0.Brain tissues were mechanically ground and lymphocytes were extracted with Percoll  . Whole bThe cells were blocked with anti-CD16/32  to reduce nonspecific binding.Antibodies for FACS included FITC-labeled anti-mouse CD4 antibody , PE-labeled anti-mouse INF-γ antibody , PE-Cy™7-labeled anti-mouse IL-4 antibody , Alexa Fluor® 647-labeled anti-mouse IL-17A antibody (BD Pharmingen Cat. No. 560184), PE-labeled anti-mouse CD25 antibody , and Alexa Fluor® 647-labeled anti-mouse Foxp3 antibody .The cells were stained with CD4, INF-γ, IL-4, and IL-17A antibodies to define Th1, Th2, and Th17 cells and CD4, CD25, and Foxp3 antibodies to define Treg cells.t test with GraphPad Prism software V9.4.0 . All hypothesis tests were conducted at a significance level of 0.05.All experiments were repeated at least three times independently. The data are expressed as mean ± standard error of the mean (SEM). The statistical analysis involved repeated measures ANOVA, one-way ANOVA, and Student’s P > 0.05). This result suggested that there was no impaired spatial learning in 3-month-old APP/PS1 mice. In comparison, 12-month-old APP/PS1 mice had significantly longer escape latencies than the matched WT mice. In male mice, the latency period of APP/PS1 mice began to be significantly longer than that of matched WT mice by the third day (P < 0.05)   . Nevertheless, no such significant differences were observed in female mice and APP/PS1 male mice . Nevertheless, in 3-month-old mice  and in 12-month-old APP/PS1 male mice, the differences in alveolar bone volume and bone microarchitecture were not evident  staining of periodontal tissue in the maxillary first molar is shown in Fig. In male mice, the expression of Th1 and Th17 cells in PBMCs was significantly upregulated in 12-month-old APP/PS1 mice, while the expression of Th2 decreased compared with that in age-matched WT mice Fig. A, C. TheIn female mice, the expression of Th1, Th17 and Treg cells in PBMCs was significantly upregulated in 12-month-old APP/PS1 mice, while the expression of Th2 cells decreased, compared with that of age-matched WT mice Fig. B, C. SimIn different age comparisons between the same sex, the expression of Th1, Th2, Th17 and Tregs was significantly higher in the brain tissue of 12-month-old male mice compared to 3-month-old males, and a similar trend could be observed in females. However, in PBMCs, there were significant sex differences in this age comparison. In the same-age comparison across sex, in both brain tissue and PBMCs, the expression of Th1 cell was greater in 3-month-old female mice than in males, while Treg cell expression was greater in males than in females. In 12-month-old APP/PS1 mice, the expression of Th1 in PBMCs was greater in males than in females, but the expression of Th2 and Treg were smaller in males than in females. In addition, the expression of Th1 and Th2 cells were greater in the brain tissue of 12-month-old APP/PS1 male mice than in females  and effector T cells  may be the underlying mechanism. However, this immune state is independent of sex and age.The association between Alzheimer’s disease and periodontitis has recently received increasing attention . WhetherThe APP/PS1 transgenic mouse is an ideal model to examine the underlying AD pathophysiology and cognitive ethology . The micP. gingivalis or molar ligation to induce periodontal bone loss and thus, the outcome cannot be explained by microbial infection. In summary, it is highly likely that alveolar bone resorption and impairment in the periodontium in APP/PS1 mice are related to the pathological process of AD.According to the published literature, an epidemiological correlation between AD pathogenesis and the loss of dental elements is supported by different cohort studies . PatientThe homeostasis of the skeletal system depends on the dynamic balance between osteogenesis by osteoblasts and bone resorption by osteoclasts . ShiftinPeriodontitis is a chronic bacterial infectious inflammatory disease in which plaque stimulates the host inflammatory response, leading to periodontal tissue damage and bone resorption. Although many studies in the last decades have focused on plaque, the major periodontal tissue destruction may be due to a series of immune inflammatory responses to bacteria . PeriodoIt is increasingly important to acknowledge sex differences in immune responses and bone homeostasis. Since 2015, the field of sex-based biology has launched several new policies to promote more consideration, reporting and analysis of sex in biomedical science to improve the rigor of research .Sex is one of the major risk factors for AD, with a higher incidence of the disease in females . OsteopoEstrogen regulates elements related to immune function and bone metabolism, which is reflected in the activation of osteoclasts by T cells. The influence of estrogen on the T-cell response is biphasic. Estrogen has been shown to affect cytokine production of both the type 1 and type 2 helper T-cell subsets . In animAge and sex are important variables to consider in evaluating periodontal bone tissue, and the cognitive impairment of APP/PS1 mice is more related to age. Cognitively impaired 12-month-old APP/PS1 female mice had more impaired periodontal tissues  compared to age- and sex-matched WT mice. In addition, immune dysregulation  was found in the brain tissue and PBMCs of APP/PS1 mice, but this alteration of immune state was not statistically related to sex or age. The imbalance in immune homeostasis may be the mechanism of the impact of AD on the destruction of periodontal bone, while this hypothesis still needs to be further confirmed in individual mice. More experiments are needed in the future to prove that there is a correlation mechanism between AD immune inflammation and periodontal conditions.Additional file 1: Figure S1. Percentage of distance in the target quadrant. A, B Difference between WT mice and APP/PS1 mice within the same age and sex. C, D Difference between 12-month-old and 3-month-old mice within the same genotype and sex. E, F Difference between males and females within the same genotype and age.Additional file 2: Figure S2. Assessment of bone microstructure in molar region by micro CT. A. The difference between 12-month-old and 3-month-old mice within the same genotype and sex. B. The difference between males and females within the same genotype and age.Additional file 3: Figure S3. CD4+ T cells in PBMCs by flow cytometry. A. The difference between 12-month-old and 3-month-old mice within the same genotype and sex. B. The difference between males and females within the same genotype and age.Additional file 4: Figure S4. CD4+ T cells in brain tissues by flow cytometry. A. The difference between 12-month-old and 3-month-old mice within the same genotype and sex. B. The difference between males and females within the same genotype and age.